牙龈卟啉单胞菌脂多糖通过TXNIP/Nlrp3炎性通路对小鼠牙周膜成纤维细胞迁移的影响

Effect of Porphyromonas Gingivalis Lipopolysaccharide on Migration of Periodontal Ligament Fibroblasts ViaTXNIP/Nlrp3 Inflammasome Pathway

目的:研究牙龈卟啉单胞菌脂多糖(Porphyromonas lipopolysaccharide,LPS-PG)对牙周膜成纤维细胞(mouse periodontal Ligament:Normal Fibroblasts,m PDLFs)增殖及迁移的影响,探讨TXNIP/Nlrp3炎性体途径在其中的作用。方法:采用不同浓度的LPS-PG刺激小鼠m PDLFs细胞不同时间,CCK-8法检测细胞增殖抑制率。然后将细胞分为对照组(培养基作用24 h)和LPS-PG组(2 M的LPS-PG作用24 h),划痕实验检测细胞迁移,ELISA法检测白细胞介素-1β(Interleukin-1β,IL-1β)和高迁移率族蛋白B1(High mobility group protein B1,HMGB1)的水平,Western Blot检测Nod样受体蛋白3(Nod-like receptor pyrin domain3,NLRP3)、凋亡相关斑点样蛋白(Apoptosis-associated speck-like protein,ASC)、活化半胱氨酸蛋白酶(cleaved-caspase-1)和硫氧还蛋白相互作用蛋白(Thioredoxin-interacting protein,TXNIP)的表达,免疫共沉淀检测以上蛋白间的相互作用。结果:与0 M的LPS-PG相比,1 M和2 M的LPS-PG可显著增加m PDLFs的细胞增殖抑制率(P<0.05)。与LPS-PG作用0 h相比,LPS-PG作用12 h、24 h和48 h均可显著增加m PDLFs的细胞增殖抑制率(P<0.05)。LPS-PG组细胞向中间’伤口’迁移的距离及细胞数量远远低于对照组。与对照组相比,LPS-PG组细胞中IL-1β和HMGB1的水平、NLRP3、cleaved-caspase-1和TXNIP蛋白表达均显著增加,且NLRP3和ASC、NLRP3和cleaved-caspase-1、TXNIP和NLRP3的蛋白互作能力显著增强(P<0.05),而两组ASC蛋白的表达无显著差异(P>0.05)。结论:LPS-PG可抑制m PDLFs细胞的增殖和迁移,其机制与Nlrp3炎性体的形成与活化有密切的关系。

Objective: To investigate the effects of lipopolysaccharide from Porphyromonas gingivalis(LPS-PG) on the proliferation and migration of periodontal ligament fibroblasts(m PDLFs), and to explore the role of TXNIP/Nlrp3 inflammasome pathway in it.Methods: Different concentrations of LPS-PG were used to stimulate mouse m PDLFs cells for different times, and the cell proliferation inhibition rate was detected by CCK-8 method. The cells were then divided into two groups: control group(stimulation with medium for24 h) and LPS-PG group(stimulation with 2 M LPS-PG for 24 h). The scratch test was used to detect cell migration, ELISA method was used to detect the level of IL-1β and HMGB1, Western Blot was used to detect the expression of NLRP3, ASC, cleaved-caspase-1 and TXNIP protein, and Co-immunoprecipitation is used to detect the interaction between the above proteins. Results: Compared with 0 M of LPS-PG, 1 M and 2 M of LPS-PG significantly increased the cell proliferation inhibition rate of m PDLFs(P<0.05). Compared with LPS-PG at 0 h, LPS-PG at 12 h, 24 h and 48 h can significantly increase the cell proliferation inhibition rate of m PDLFs(P<0.05). The migration distance and the number of cells in the LPS-PG group to the middle ’wound’ were much lower than those in the control group.Compared with the control group, the levels of IL-1β and HMGB1 and the expression of NLRP3, cleaved-caspase-1 and TXNIP protein in the LPS-PG group were significantly increased(P< 0.05), and the protein interaction ability of NLRP3 and ASC, NLRP3 and cleaved-caspase-1, TXNIP and NLRP3 was significantly enhanced(P<0.05), but there was no significant difference in the expression of ASC protein between the two groups(P>0.05). Conclusion: LPS-PG can inhibit the proliferation and migration of m PDLFs cells, and its mechanism is closely related to the formation and activation of Nlrp3 inflammasome.